Abstract
The adipose-derived hormone leptin is important for regulating reproductive processes, including puberty onset. Leptin mediates its effects via a multitude of signalling pathways, and the canonical Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway is the most well-characterised leptin receptor (LepR) signalling pathway. Neural STAT3 deletion is known to cause obesity; however, its reproductive influence is less understood.
Previous data suggested that STAT3 signalling might be unnecessary for puberty timing and reproduction. However, these studies utilised mice on a C57BL/6J genetic background, a strain where reproductive capacity is barely affected by metabolic challenges. This prompted a re- evaluation using a more suitable background strain, DBA/2J, which is susceptible to reproductive impairments and metabolic challenges. Mice with STAT3 deletion in LepR- expressing cells on C57BL/6J and DBA/2J backgrounds showed no differences in puberty onset or adult fertility compared to controls. Additionally, no or minimal metabolic phenotype was noted. Surprisingly, there were no differences between the background strains. Immunohistochemical analysis of arcuate (ARC) brain sections revealed no notable loss of STAT3 activation in response to a leptin injection in the knockout groups. This suggests that the LepR Cre might be a weak driver of Cre-mediated excision. Thus, it was concluded that the choice of Cre-line used is more critical than the genetic background of the mice.
To further assess the requirement of specific leptin signalling pathways in mediating leptin’s reproductive effects, transgenic mice with neuronal knockouts were created using the Cre-loxP system. CamKinaseIIα-Cre mice were crossed with either STAT3, mammalian target of rapamycin (mTOR), or extracellular signal-related kinase 2 (ERK2) floxed mice. Puberty onset was assessed post-weaning by examining genitalia development. Reproductive cyclicity and organ weights were measured in adults. Metabolic effects were evaluated through body weight, abdominal fat, and fasting glucose measurements. Immunohistochemical brain tissue analysis evaluated cellular responses to leptin for STAT3, ERK2, and mTOR; the activated forms of these signalling molecules were abundant in the ARC of intact control mice but nearly absent in knockout mice. Mice with ERK2 or mTOR knockouts showed no reproductive or metabolic deficits compared to controls. In marked contrast, neuronal STAT3 knockout mice showed significantly increased body weight by five weeks of age and a six-fold increase in abdominal adiposity as adults compared to controls. Neuronal STAT3 knockout mice also exhibited more than a two-fold elevation in fasting glucose levels. When puberty was assessed, it was observed that males had a significant five-day delay in the age of preputial separation. In contrast, females had a significant seven-day delay in vaginal opening and a nine-day delay in first estrus. Additionally, STAT3 knockout females showed pronounced acyclicity, while subfertility and regressed reproductive organ weights were observed in both sexes. These data indicate that neuronal STAT3 is critical for correctly timed puberty and subsequent fertility in male and female mice, whereas mTOR and ERK2 are not essential. These results prompted a re-evaluation of previous conclusions about the role of STAT3 from experiments using LepR-Cre mice.
To determine whether agouti-related peptide (AgRP) neurons could be partially responsible for mediating the delays in puberty onset observed in neuronal STAT3-deficient mice, STAT3 deletion was targeted to AgRP neurons, a leptin-responsive population necessary for normal pubertal timing and reproduction. AgRP-Cre mice were crossed with STAT3 floxed mice to achieve STAT3 knockout in AgRP neurons. Similarly to the previous experiments, puberty onset was assessed post-weaning, and reproductive cyclicity, organ weights, and fecundity were measured in adults. Metabolic assessments included weekly body weight and composition analysis, abdominal adiposity, insulin tolerance, food intake, and activity monitoring. Brain tissue analysis evaluated the colocalisation of fluorescent STAT3 activation and AgRP. Female mice with STAT3 knockout in AgRP neurons showed significantly increased body weight by three weeks of age and a significant three-day delay in first estrus. Additionally, females with STAT3 deletion in AgRP neurons exhibited a significant delay in the time from mating to the first litter. No reproductive or metabolic deficits were observed in males with STAT3 deletion in AgRP neurons compared to controls. The requirements for STAT3 in AgRP neurons precisely mirrored our group's earlier characterisation of AgRP requirements for leptin receptors. This supports the role of STAT3 signalling in AgRP neurons as a key mediator of leptin’s reproductive control.
To contextualise these neuronal knockouts, mice with novel LepR mutations resulting from single amino acid substitutions (A63C and P1018S) were used to assess leptin-induced signalling pathways. These mice demonstrated a significant delay in puberty onset in males and females, mimicking the effects originally characterised in davisdale ewes. The brains of these mice offered a valuable opportunity to investigate potential deficits in LepR-signalling within a context that closely mirrors real-world conditions. Immunohistochemistry was used to assess leptin-induced STAT3, ERK1/2, and mTOR signalling in response to a submaximal leptin challenge to reveal subtle variations in leptin signalling. Interestingly, the results demonstrated that neither STAT3, ERK1/2, nor mTOR appeared downregulated in response to the A63C or P1018S LepR mutations. These findings indicate that neither the compromise nor compensation of leptin signalling in A63C and P1018S mice involves detectable changes in the levels of STAT3, ERK1/2, or mTOR signalling, suggesting that other pathways might be involved (or the type of analysis used lacked the fidelity to detect subtle alterations in leptin intensity).
Overall, this work underscores the intricate role of leptin in regulating fertility, with the STAT3 pathway emerging as a crucial mechanism through which leptin influences reproduction, partly mediated by AgRP neurons. The research still allows for leptin signalling involving multiple redundant pathways to maintain reproductive function, rather than relying on a single pathway or specific neuronal population, but now STAT3 appears to be key amongst these. These findings offer new insights into the pathophysiology of reproduction under altered metabolic conditions, enhancing our understanding of how leptin and its associated pathways regulate the onset of puberty and fertility.